The Extracytoplasmic Domain of the Mycobacteriumtuberculosis Ser/Thr Kinase PknB Binds SpecificMuropeptides and Is Required for PknB LocalizationMushtaq Mir1, Jinkeng Asong2, Xiuru Li2, Jessica Cardot2, Geert-Jan Boons2, Robert N. Husson1*

1 Division of Infectious Diseases, Children’s Hospital Boston and Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Chemistry and

the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States of America

Abstract

The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology inthis organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thrkinase associated (PASTA) domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensivelibrary of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in amanner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and onthe presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes stronglyto the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrastto strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a syntheticmuropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitationof dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenouspeptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likelythrough binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest amodel in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.

Citation: Mir M, Asong J, Li X, Cardot J, Boons G-J, et al. (2011) The Extracytoplasmic Domain of the Mycobacterium tuberculosis Ser/Thr Kinase PknB Binds SpecificMuropeptides and Is Required for PknB Localization. PLoS Pathog 7(7): e1002182. doi:10.1371/journal.ppat.1002182

Editor: William R. Bishai, Johns Hopkins School of Medicine, United States of America

Received December 7, 2010; Accepted June 12, 2011; Published July 28, 2011

Copyright: � 2011 Mir et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by National Institutes of Health Grants R21AI062275 and R01AI059702 to RNH, and 2R01GM061761 to G-JB. The funders hadno role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: robert.husson@childrens.harvard.edu

Introduction

Bacterial cell growth and cell division are highly regulated

processes, requiring the coordination of multiple activities within the

cell. DNA replication and chromosome segregation for example,

must occur at the correct time and in the correct location, and be

coordinated with septum formation and cytokinesis. The molecules

involved in septum formation and the sequence in which they are

recruited to the division site have been the subject of intense

investigation in the model organisms Bacillus subtilis and Escherichia

coli, and the identities and functions of many bacterial cell division

proteins have been elucidated [1,2]. In addition to divisome

assembly and DNA segregation, bacterial growth and cell division

require remodeling of the peptidoglycan (PGN) mesh that forms the

cell wall [3]. The enzymes and the sequence of reactions involved in

cell wall synthesis are relatively well understood as are the enzymatic

activities of many of the PGN hydrolases that can degrade this

polymer [4,5]. In the model organism B. subtilis, the mechanisms by

which cell wall hydrolases are regulated to achieve morphogenesis

are at least partially understood [6]. In other bacteria, including the

slow growing actinomycete Mycobacterium tuberculosis, less is known

about the regulation of PGN synthesis and hydrolysis, how these

opposing processes are balanced, and how they are coordinated

with other cell processes in growing and dividing vs. non-growing

dormant cells.

Because of the apparent ability of M. tuberculosis to become

dormant in the human host, leading to asymptomatic latent

infection, there has been great interest in understanding how cell

growth and cell division are regulated in this organism [7]. A

longstanding observation that ‘‘spent’’ or ‘‘conditioned’’ medium,

i.e. filter-sterilized supernatant from bacterial cultures grown in

liquid medium, is able to stimulate growth of dormant cells, led to

the identification of a resuscitation promoting factor (Rpf) by

purifying from spent medium a component that was able to

stimulate growth of the actinomycete Micrococus luteus [8]. Rpf is

small protein that has homologues in other actinobacteria, including

M. tuberculosis, which has five rpf genes [9]. Functional studies of

these genes in M. tuberculosis have shown that individually they are

not required for resuscitation of dormant M. tuberculosis cells and

single rpf mutant strains do not have other growth or morphologic

phenotypes. When two or more rpf genes are inactivated, however,

growth or resuscitation defects are observed [10,11,12]. The recent

demonstration that the Rpf’s are PGN hydrolases suggests that

growth stimulation of dormant cells may result from the enzymatic

activity of these secreted proteins, possibly through alterations in

PGN structure or through the interaction of PGN degradation

products with the bacterial cell surface [13].

A domain found to occur in the extracytoplasmic regions of

penicillin binding proteins and serine/threonine kinases (PASTA

domain) was identified by bioinformatic analysis and predicted to

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bind to the stem peptide of un-crosslinked PGN precursors, based

on the structure of the PASTA-containing penicillin binding

protein PBP2X of Streptococcus pneumoniae bound to a cephalosporin

antibiotic [14]. Recently the PASTA domain of a Ser/Thr kinase

of B. subtilis was shown to bind both intact and hydrolyzed PGN

[15]. Incubation of B. subtilis spores with PGN stimulated spore

germination and increased Ser/Thr phosphorylation. Some

specificity with respect to the source of PGN and these functional

effects was observed, suggesting a preference for meso-diamino-

pimelic acid (m-DAP)-containing PGN in stimulating spore

germination in this organism.

The M. tuberculosis genome encodes two proteins that contain

PASTA domains, the Ser/Thr protein kinase PknB (Rv0014c)

whose extracytoplasmic region comprises four PASTA domains,

and the bifunctional penicillin binding protein PBP2 (PonA2,

Rv3682), which has a single PASTA domain at the extreme

carboxy-terminus of the protein distal to the extracytoplasmic

transpeptidase and transglycosylase-containing regions [16]. In

this work we investigated the quantitative binding of a series of

synthetic muropeptides to the extracytoplasmic region of PknB.

We identified specific features of these molecules that are required

for high affinity binding, and investigated the functional effects of

these compounds in vivo on mycobacterial growth, morphology

and the localization of PknB. We determined that PknB is strongly

localized to septum and less strongly to the cell poles, the sites of

active PGN synthesis in mycobacteria, and that the PASTA

domains of PknB are required for its localization.

Results

Binding of PGN fragments to the extracytoplasmicdomain of PknB

The region of pknB that encodes the extracytoplasmic domain of

PknB (ED-PknB) was amplified by PCR, cloned and ED-PknB was

expressed in Escherichia coli as an N-terminal Glutathione-S-

transferase (GST) fusion protein. The ED-PknB comprises 4

PASTA motifs that share limited sequence similarity aside from

the key residues that define the motif (Figure S1). Soluble

recombinant GST-ED-PknB was affinity purified to .95% purity

and after removal of the GST tag was used in subsequent binding

experiments (Figure S2).

A series of PGN fragments (muropeptides) were synthesized as

tri-, tetra- and penta-peptides linked to N-acetylmuramic acid

(MurNAc) or as unlinked peptides. Amino acids characteristic of

PGN stem peptides from Gram-positive bacteria, Gram-negative

bacteria or actinobacteria were incorporated into different

compounds. Modifications of amino acid side chains that

correspond to PGN modifications that are found in vivo were also

included in the compound series (Figure 1). These compounds

were then used in surface plasmon resonance (Biacore) experi-

ments to measure binding affinities of the muropeptides to ED-

PknB. To obtain kinetic and thermodynamic parameters, a range

of compound concentrations was assayed and kinetic analysis was

performed using Biacore Software. An example of a set of

sensorgrams for a compound with a relatively low KD is shown in

Figure 2. Sensorgrams for the other compounds tested are shown

in Figure S3. Table S1 shows detailed kinetic parameters obtained

from these experiments.

As shown in Table 1, these experiments demonstrated moder-

ately strong binding of several PGN fragments that have DAP at the

third position of the stem peptide. N-acetylation of the amino group

of DAP as in compound 6e (MTrP-DAP (amide/acid) NHAc),

which is designed to mimic branching of the PGN subunits within

the PGN polymeric structure, resulted in a six-fold decrease in

binding compared to compound 6a. The MurNAc-pentapeptide,

compound 7, corresponding to newly synthesized PGN prior to

remodeling, bound strongly though about two-fold less than the

corresponding MurNAc-tetrapeptide (6a).

In addition to preference for DAP at the third position of the

stem peptide, another clear result of these experiments is the

requirement for amidation of D-isoglutamate (D-iGlu) to D-

isoglutamine (D-iGln) at the second position, in order to achieve

high affinity binding. Compound 6a, which contains both D-iGln

and DAP at the second and third positions, respectively, exhibited

the highest affinity, while compound 6d, which is identical except

for D-iGlu at the second position, bound four-fold less strongly.

Similarly, compound 6c, which also bound with a relatively high

affinity (KD = 15 mM), contains D-iGln together with amidation of

the carboxyl group of DAP. In contrast, a similar compound, (6b),

that has D-iGlu at the second position instead of D-iGln did not

show measurable binding. The importance of this residue is

further underscored by the finding that among the Lys-containing

compounds, the only one that showed detectable interaction was

compound 2a, the muramyl tetrapeptide incorporating a D-iGln

moiety. While the data indicate a preference for DAP at the third

position, the e-carboxylic acid group that is a major feature that

distinguishes DAP from Lys is not an essential requirement for

binding. To determine whether the MurNAc moiety was

important for binding, compounds 4 and 8, pentapeptides not

linked to MurNAc and containing either Lys or DAP at the third

position, respectively, were tested. Neither compound showed

significant interaction, indicating an important contribution of

MurNAc in binding to the PknB PASTA domains.

Muropeptides stimulate resuscitation of dormant M.tuberculosis cells

The Rpf’s have been shown to have PGN hydrolytic activity,

and are thought to cleave the ß-1–4 glycosidic linkage between N-

acetylmuramic acid and N-acetylglucosamine [13]. This muralytic

activity has been shown to be essential for the resuscitation activity

of these Rpf proteins, but the mechanism remains uncertain. To

determine whether muropeptides that bind to the PASTA

Author Summary

Regulation of growth by Mycobacterium tuberculosis isimportant in the pathogenesis of tuberculosis (TB),including asymptomatic latent TB infection and active TBdisease. The M. tuberculosis kinase PknB regulates cellgrowth and cell division by phosphorylating proteinsinvolved in these processes to modify their function. Theactivity of PknB is thought to respond to extracellularstimuli by binding specific molecules with its extracyto-plasmic domain. In this work we show that cell wallfragments bind to this domain, and that strong bindingrequires that these interacting molecules have specificmolecular features. We demonstrate that a peptidoglycanfragment that binds strongly can stimulate growth ofdormant bacteria, but that it does not affect growth ofnon-dormant bacteria. We also show that PknB localizes tothe site of cell division and to the growing tip of thebacterium, where cell wall synthesis and degradationoccur, and that the extracytoplasmic domain is requiredfor this localization. These findings indicate that a majorfunction of the extracytoplasmic domain of PknB is toplace it at the sites of cell wall turnover, and suggest amodel by which PknB can regulate growth and celldivision, and thereby contribute to the pathogenesis of TB.

Muropeptides Bind and Localize PknB

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Figure 1. Structures of synthetic muropeptides used in the binding and phenotypic assays. Lys-containing compounds are typical ofGram-positive bacteria and DAP-containing compounds are typical of Gram-negative bacteria and Actinomycetes, including mycobacteria. Variationsin substituents are indicated in red, and the specific variations are listed immediately below the structure.doi:10.1371/journal.ppat.1002182.g001

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domains of ED-PknB can stimulate resuscitation of dormant M.

tuberculosis cells, we utilized an established M. tuberculosis dormancy

and resuscitation model [17]. In this assay, M. tuberculosis cells are

incubated under hypoxic conditions for several months, at which

point the number of cells capable of resuming growth in liquid

culture is markedly decreased. In this assay, addition of sterile

spent medium ‘‘resuscitates’’ dormant cells, leading to an increase

in the number of cells that can grow on solid or in liquid medium.

In two independent experiments, we took M. tuberculosis

stationary phase cultures that had been incubated under hypoxic

conditions for 6 or 9 months, and performed this resuscitation

assay. In addition to cells incubated in Sauton’s medium alone,

cells were incubated with a synthetic muropeptide with a high

affinity for ED-PknB (6c in Figure 1), a muropeptide with low

affinity for ED-PknB (3b in Figure 1), or with sterile conditioned

medium as a positive control. The muropeptides were used at a

concentration of 10 times the KD of the high affinity compound as

determined in the SPR experiments. Using most probable number

analysis [18], which has been used to analyze results from this

assay, we observed three and nine-fold increases in the viability of

cells that were incubated with the high affinity muropeptide in the

two independent experiments. No increase in viability was

observed for cells incubated with the low affinity peptide. The

cells incubated with sterile spent medium showed a much stronger

resuscitation phenotype, with 14 and 100-fold increased viability

relative to the cells incubated in fresh medium alone (Table 2).

The original identification of Rpf in M. luteus was based on the

observation that stationary phase cells show decreased viability

when plated or diluted to low density in liquid medium, but that

addition of sterile conditioned medium stimulates growth [8]. A

similar phenomenon is observed when mycobacteria are inocu-

lated at low density. To determine whether synthetic muropeptides

stimulate growth when stationary phase cells are inoculated at low

density, cells from cultures of M. tuberculosis (O.D600 of 2.4–3.6)

were washed and diluted 10,000-fold in minimal medium with or

without the addition of the high or low affinity muropeptide. As

shown in Figure 3, no growth stimulation by either muropeptide

was observed in this assay. In contrast, strong growth stimulation

by conditioned medium was observed.

PknB is present in the cell envelope of M. tuberculosisBased on its sequence, PknB is predicted to have a single

transmembrane segment, with an intracellular kinase domain and

an extracytoplasmic region that incorporates the four PASTA

domains [16]. To determine whether PknB is a membrane protein

and in which subcellular fraction(s) PknB is located, we performed

immunoblotting with a PknB-specific monoclonal antibody. As a

Figure 2. Sensorgrams of a compound with relatively highbinding affinity for ED-PknB. The sensorgrams show the simulta-neous concentration-dependent kinetic analysis of two-fold serialdilutions of MTrP-DAP (amide/acid) (Compound 6c in Figure 1) atconcentrations from 1.56 mM to 100 mM. ED-PknB was bound to thesensor chip and at time 0 the muropeptide was flowed over the chip,followed by a buffer only dissociation step, as described in the Materialsand Methods section. Positive deflection of the curve indicates bindingin RU (resonance units). The primary data are shown in red. The datawere fitted with a two-state binding model (black lines). Thecorresponding residual values, which are the signal remaining afterthe data are fitted to the kinetic model, are plotted below thesensorgrams.doi:10.1371/journal.ppat.1002182.g002

Table 1. Affinity of synthetic muropeptides for theextracytoplasmic domain of M. tuberculosis PknB.

Analyte KD (mM)

MTP-Lys (amide) 1 .500

MTrP-Lys (amide) 2a 21.5

MTrP-Lys (amide) NHAc 2b .500

MTrP-Lys (Gly) 2c . 500

MPP-Lys (D-Ala) 3a . 500

MPP-Lys (Gly) 3b .500

Peptide 4 (amide) . 500

MTP-DAP (amide/acid) 5 21.8

MTrP-DAP (amide/acid) 6a 12.7

MTrP-DAP (acid/amide) 6b .100

MTrP-DAP (amide/amide) 6c 14.9

MTrP-DAP (acid/acid) 6d 53.6

MTrP-DAP(amide/acid)NHAc 6e 73.8

MPP-DAP (amide/acid) 7 25.1

Peptide 8 (amide/amide) .500

Recombinant ED-PknB was immobilized on NHS-activated groups of a CM-5sensor chip surface (5,000 RU) and titration experiments were performed withthe synthetic compounds 1–8 (Figure 1). The binding constants of allcompounds were determined by fitting the data using a two-state bindingmodel. Kinetic binding parameters and the sensorgrams for the kinetic analysesare presented in Table S1 and Figure S3. MTP, muramyl-tripeptide; MTrP,muramyl-tetra peptide; MPP, muramyl-pentapeptide.doi:10.1371/journal.ppat.1002182.t001

Table 2. Resuscitation of dormant M. tuberculosis cultures.

Additive to Culture Medium Fold increase*

Experiment 1:6 month old dormant culture

MTrP-DAP (amide/amide) (6c) 9

MPP-Lys (Gly) (3b) 0.9

50% spent medium 100

Experiment 2:9 month old dormant culture

MTrP-DAP (amide/amide) (6c) 3

MPP-Lys (Gly) (3b) 1

50% spent medium 13.6

*Fold increase in viable cell number relative to cultures grown in Sauton’smedium without additive.doi:10.1371/journal.ppat.1002182.t002

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control, we probed these subcellular fractions with an antibody to

the membrane protein PknA, which like PknB has a single

transmembrane segment, but which has a small extracytoplasmic

region that is not known to interact with cell wall components. We

found that PknB does, as predicted, localize to the membrane

fraction of the cell (Figure 4). An even stronger signal was seen in

the cell wall fraction, further confirming the association of PknB

with the cell envelope. The PknA antibody gave equally strong

signals from the membrane and cell wall fractions. These results

demonstrate that PknB is a membrane protein and that membrane

is present in the cell wall fractions used in these experiments.

PknB localizes to the septum and poles, and theextracytoplasmic domain is required for proper PknBlocalization

A construct designed to express a PknB-RFP fusion protein, in

which RFP is fused to the amino terminus of PknB, was introduced

into wild type M. smegmatis. Additional constructs, in which RFP is

fused a) to the PknB kinase domain, intracellular juxtamembrane

sequence and transmembrane segment, but which lacks the

extracytoplasmic domain, and b) to the membrane and ED-PknB

regions but which lacks the intracellular linker and kinase

domains, were also introduced into wild type M. smegmatis. Cells

were grown to early log phase, expression of the fusion protein was

induced, and the cells were examined using fluorescence

microscopy (Figure 5). Cells expressing the full-length PknB-RFP

fusion showed strong localization of this protein to the mid-cell

and symmetrical, less intense localization to both cell poles. In

contrast, in cells expressing the fusion that lacks the extracyto-

plasmic domain containing the PASTA domains, foci of

fluorescence were visible at discrete sites along the length of the

cell. While in some cells there appears to be increased signal at the

poles, we did not observe clear mid-cell localization in cells

expressing this construct. To confirm that these foci are not

cytoplasmic aggregates, we prepared subcellular fractions of these

cells and confirmed that the large majority of this protein is present

in the cell membrane and cell wall fractions (Figure S4). Cells

expressing the ED-PknB-RFP fusion lacking the intracellular

linker and kinase domains showed clear localization to the mid-cell

but minimal signal from the poles. This result indicates that the

extracytoplasmic PASTA domains are required for proper

localization of PknB to the mid-cell and likely to the cell poles

and suggests that the intracellular linker-kinase region makes a

contribution to localization at the cell poles. To verify these

findings, we performed additional imaging of live cells (Figure S5

and Protocol S1), which demonstrates the same localization

patterns observed with the fixed cell preparations.

To determine whether diffusible, non-localized muropeptides

might bind ED-PknB and disrupt PknB localization, we incubated

M. smegmatis for 8 hours with the high affinity muropeptide used in

the resuscitation experiments. No change in the morphology of

wild type bacteria were observed, and in the strain expressing the

Figure 3. Growth stimulation assay of low inoculum cultures ofM. tuberculosis. M. tuberculosis cells were grown in Sauton’s mediumalone or in medium supplemented with a synthetic muropeptide at aconcentration 10 times the KD, of the high affinity muropeptide, or with50% (v/v) sterile conditioned (spent) medium. Cells were grown tostationary phase, diluted and inoculated into medium containingalamar blue and grown at 37uC, with measurement of fluorescence at595 nm at serial time points. A. Growth curves for MPP-Lys (Compound3b in Figure 1). B. Growth curves for MTrp-DAP (Compound 6c inFigure 1). N, Sauton’s medium. &, Sauton’s medium plus syntheticmuropeptide. m, Sauton’s medium plus spent medium.doi:10.1371/journal.ppat.1002182.g003

Figure 4. Subcellular localization of M. tuberculosis PknB. Toppanel: Immunoblot of subcellular fractions of M. tuberculosis, probedwith a mouse monoclonal antibody that recognizes M. tuberculosis ED-PknB. Bottom panel: immunoblot in which the same fractions wereprobed with rabbit polyclonal sera raised against M. tuberculosismembrane protein PknA. CM, membrane fraction; CY, cytoplasmicfraction; CW, cell wall fraction; CF, culture filtrate fraction.doi:10.1371/journal.ppat.1002182.g004

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PknB-RFP fusion protein the RFP signal remained localized to the

septum and poles (not shown).

Discussion

In this work we report three major findings. First, we

demonstrated that muropeptides bind to the extracytoplasmic

region of PknB, which contains four PASTA domains, and defined

molecular requirements for ligand binding. These requirements

include both specific residues at the second and third positions in

the stem peptide, and the presence of the sugar moiety (MurNAc)

linked to the amino-terminal residue of the peptide. Using an

extensive series of chemically synthesized compounds, we found

moderately high affinity binding by muropeptides that contain

DAP at the third position of the stem peptide, in which the D-iGlu

at the second position is amidated to D-iGln. The preference for

DAP is consistent with the predominant structure of the stem

peptide of mycobacteria, where DAP is present at this position, in

contrast to most Gram-positive organisms in which Lys occurs at

this position. D-iGln at the second position has been reported to be

predominant in M. tuberculosis PGN, however D-iGlu is present in

a minority of stem peptides [19,20]. Whether synthesis of PGN

incorporating D-iGlu vs. D-iGln is site- or growth-stage specific in

M. tuberculosis is not known. The markedly stronger binding of

compounds containing D-iGln suggests that variation in the

structure of PGN stem peptides may affect binding by ED-PknB in

vivo, with potentially important physiologic effects. A recent paper

examining stimulation of B. subtilis spore germination using

synthetic muropeptides confirmed prior results using purified

native PGN in showing the importance of DAP at the third

position of the stem peptide for this phenotype in this species [21].

In this assay the presence of N-acetylglucosamine linked to

MurNAc was also required for potent activity.

A second finding of this work is that PknB localizes strongly to

the mid-cell and less strongly to the cell poles of mycobacteria, the

sites of active PGN synthesis and hydrolysis in these organisms

[22]. Our results with RFP fusions to full-length PknB and to

separate domains of this protein in M. smegmatis demonstrate that

the PASTA motif-containing extracytoplasmic domain of PknB is

required for its localization to the mid-cell. We attempted to

perform a similar experiment with full-length PknB-RFP in M.

tuberculosis, however we were unable to obtain consistent expression

of the fusion protein. We observed fluorescence in a minority of

cells, which was highly variable from cell to cell, and we observed

markedly abnormal morphology of many cells, suggesting severe

toxicity of pknB overexpression, as previously described [23].

Despite these limitations, we were able to see similar localization of

full-length PknB in a minority of rod-shaped cells expressing the

pknB-rfp fusion (data not shown).

In the context of our in vitro binding results, these data suggest

that binding of PGN fragments by its extracytoplasmic domain is

critical for PknB localization to the mid-cell and possibly to the

poles. This result, together with the finding that PknB is found in

the cell wall and membrane fractions of M. tuberculosis lysates,

suggests that the PASTA motifs of PknB bind endogenous cell wall

Figure 5. Localization of PknB to sites of peptidoglycanturnover in the mycobacterial cell. A. Schematic representationof domain organization of M. tuberculosis PknB, including the kinase

domain (residues 1–274; the juxtamembrane linker (residues 275–331),the transmembrane segment (residues 332–354) and the extracyto-plasmic PASTA domains (residues 355–626). B–I. Flourescence (B, D, Fand H) and DIC (C, E, G and I) images of M. smegmatis expressing RFPfused to full-length PknB (B and C), PknB lacking the extracytoplasmicdomain (D and E), PknB lacking the intracellular kinase domain andlinker (F and G) and unfused RFP (H and I). Arrowheads in panel D pointto focal RFP signals along the length of the bacillus. Bar = 1 mm.doi:10.1371/journal.ppat.1002182.g005

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or membrane-anchored PGN precursors and/or PGN hydrolysis

fragments produced at the septum and poles of the cell. The

finding that incubation of growing cells with a high affinity

muropeptide had no effect on PknB localization is consistent with

this model, and suggests that exogenous muropeptides may not be

able to penetrate the complex, lipid-rich mycobacterial cell

envelope to reach the PknB PASTA domains at the surface of

the cytoplasmic membrane. Because both de novo synthesized PGN

precursors and PGN hydrolysis products are likely to be localized

at the septum and the poles, our data do not indicate which of

these are the major PknB PASTA ligands in vivo.

The third important finding of this work is that, in contrast to

spent medium, which strongly stimulated both growth of non-

dormant M. tuberculosis cells and resuscitation of dormant cells, a

muropeptide with relatively high affinity for the PASTA domains

of ED-PknB did not stimulate M. tuberculosis growth and had only a

modest effect on resuscitation. This result suggests that while

muropeptide binding may play a role in resuscitation of M.

tuberculosis, other factors present in spent medium may be more

important in stimulating M. tuberculosis growth. In this regard, D-

amino acids present in conditioned medium have recently been

shown to be a potent growth stimulus for Vibrio cholerae and to play

a key role in biofilm disruption leading to resumption planktonic

growth in B. subtilis [24,25]. Alternatively, PknB may require a

different muropeptide ligand, e.g. a disaccharide muropeptide or a

multivalent muropeptide, or higher concentrations of these

ligands, which may be present in vivo, for greater stimulation of

growth or resuscitation.

The first structure of a PASTA domain was determined as part

of the structure of PBP2x from S. pneumoniae bound to a

cephalosporin antibiotic [26]. In this structure two PASTA

domains interacted to form a compact globular domain. In recent

work, the structure of the PASTA motifs of M. tuberculosis ED-

PknB was determined using NMR and small angle X-ray

scattering [27]. While the individual folds of each PASTA domain

were similar to those of the PBP2x PASTA domains, the four

PASTA domains of PknB are organized as a linear molecule,

which is maintained with what the authors termed a ß9/ß99 brace

that prevents interactions between the individual PASTA domains

of a single molecule of PknB. A previous structure of the PknB

intracellular domain demonstrated the presence of a highly flexible

intracellular juxtamembrane segment linking the transmembrane

segment to the intracellular kinase domain, indicating that ligand

binding resulting in transmembrane propagation of conforma-

tional changes leading to PknB activation is unlikely [28].

Based on the PknB PASTAs structure, a model was proposed in

which binding of a single ligand to two molecules of PknB would

result in dimerization of the extracytoplasmic domains, which

would then cause dimerization of the intracellular kinase domains,

resulting in kinase activation [27]. Our data showing relatively

high affinity binding of muropeptide monomers, however, suggest

an alternative model by which muropeptide binding to ED-PknB

could lead to localization and activation of this kinase. In this

model, at sites of active PGN hydrolysis and synthesis, i.e. the

septum and the cell poles, local concentrations of PGN precursors

and PGN hydrolysis products will be high, and binding of these

ligands by ED-PknB would result in the septal and polar

localization of PknB that we observed. The recruitment of PknB

to these sites will consequently result in high concentrations of the

intracellular kinase domain, leading to the dimerization that

results in kinase activation [28,29,30]. PknB activation will then

lead to phosphorylation of protein substrates, resulting in

regulation of cell division and cell wall synthesis (Figure 6). In

this model, there is no requirement for binding of a single

muropeptide by PASTA domains from two PknB molecules, or for

muropeptides that diffuse from a distance, to achieve PknB

localization and activation.

In summary, we have demonstrated sequence-specific binding

of muropeptides to the PASTA domain-containing extracytoplas-

mic region of M. tuberculosis PknB, and that the presence of the

PASTA domains is required for localization of PknB to sites of

PGN turnover. In the context of our phenotypic data and the

finding that peptides that bind with high affinity have peptide

sequences characteristic of M. tuberculosis PGN, our results suggest

that in M. tuberculosis, the PknB PASTAs bind to PGN precursors

or fragments resulting from local PGN synthesis and/or hydrolysis

at the mid-cell and poles. This PASTA domain-mediated

localization provides a mechanism by which PknB and the co-

regulated kinase PknA can regulate cell division and PGN

Figure 6. Model of PknB localization and activation byinteraction of its extracytoplasmic domain with peptidoglycanfragments. In this model, the extracytoplasmic PASTA domains ofPknB bind PGN precursors and/or hydrolysis products produced at themid-cell and poles, the two sites where active PGN synthesis anddegradation occur in mycobacteria. This interaction leads to localizationof PknB at the theses sites, and to PknB activation by dimerization ofthe intracellular kinase domains. Both linear and non-linear forms of theextracytoplasmic domain are shown to allow for possible changesfollowing ligand binding. RPF, resuscitation promoting factor.doi:10.1371/journal.ppat.1002182.g006

Muropeptides Bind and Localize PknB

PLoS Pathogens | www.plospathogens.org 7 July 2011 | Volume 7 | Issue 7 | e1002182

turnover by reversible phosphorylation of proteins involved in

these processes, several of which have been shown to be PknA or

PknB substrates and to localize to these sites [22,23,31,32,33].

Materials and Methods

Strains, media, and recombinant plasmid constructionand protein production

Escherichia coli TOP10 (Invitrogen) was used for cloning and was

grown in LB broth. E. coli BL21 (DE3) (Stratagene) was used for

expression of recombinant ED-PknB. M. tuberculosis H37Rv or M.

smegmatis mc2-155 were grown at 37uC in Middlebrook 7H9 liquid

medium (Difco) supplemented with albumin-dextrose complex

(ADC), 0.2% Glycerol and 0.05% Tween 80, except for

resuscitation experiments where M. tuberculosis was grown in

Sauton’s medium (Difco). Kanamycin (50 mg ml21) or ampicillin

(100 mg ml21) was added to culture media or agar when

appropriate.

For expression and purification of ED-PknB, the nucleotide

sequence encoding PknB from Gly354 to Gln626 was PCR-

amplified from genomic DNA of M. tuberculosis H37Rv and cloned

in pGEX-4T-3 (GE Healthcare) for expression as a glutathione-S-

transferase (GS) fusion protein. Recombinant GST-ED- PknB was

affinity purified to .95% homogeneity using immobilized

glutathione agarose (Pierce) (Figure S2). To cleave the GST from

the fusion protein, the thrombin CleanCleave kit (Sigma) was

used. In brief 900 mg of purified recombinant ED-PknB-GST was

incubated with 100 mL of 50% (v/v) suspension of thrombin

agarose for 1 hr at room temperature. After centrifugation the

supernatant containing ED-PknB and free GST was incubated

with 500 mL of 50% (v/v) suspension of Glutathione-agarose for

15 min. After centrifugation the supernatant containing ED-PknB

was collected. For SPR analysis the supernatant was dialyzed

against phosphate buffered saline (PBS) pH 7.4 prior to use.

For localization of PknB or PknB lacking the extracytoplasmic

domain (PknBDED) in wild type M. smegmatis, the full length pknB

gene or the nucleotide sequence encoding the region from Met1 to

Gly354 of PknB (pknBDED) respectively, were PCR-amplified from

genomic DNA of M. tuberculosis H37Rv. Overlap PCR amplifica-

tion of the above PCR products was performed with the PCR

product of the red fluorescent protein (rfp) gene using a forward

primer annealing to the 59 region of rfp and a reverse primer

annealing to the 39 region of pknB or PknBDED to obtain the PCR

products rfp-pknB or rfp-pknBDED. A PacI site was introduced

between rfp and pknB. The fusion PCR products were cloned into

the integrating vector pMV306-pacet downstream of the inducible

acetamide promoter at NdeI and XbaI sites to obtain pMV306-

pacet-rfp-pknB or pMV306-pacet-rfp- pknBDED. To obtain recombi-

nant clone pMV306-pacet-rfp-pknBDKD expressing RFP linked to

transmembrane and extracellular domains of PknB (ED-PknB-

RFP) the pknB gene of the clone pMV306-pacet-rfp-pknB was

replaced with the nucleotide sequence encoding Ile326 - Gln626

using PacI and XbaI sites. Cloned DNA was sequenced to verify

the absence of mutations. A mycobacterial replicating vector that

constitutively expresses RFP was a gift from Eric Rubin.

Chemical synthesis of PGN fragmentsCompounds were synthesized using classical fluorenylmethoxycar-

bonyl (Fmoc) chemistry and standard manual solid-phase peptide

synthesis techniques as previously described [34,35,36,37,38,39]. In the

preparation of the peptide portion of the compounds, Sieber Amide

resin was swelled in dimethylformamide (DMF) for 45 min and then

treated with piperidine in DMF. After a reaction time of 30 min, the

solvents were removed by filtration and the resin washed with DMF,

and then treated with Fmoc-linked amino acid building blocks. This

generated PGN partial structures with N-termini.

Compound 8 was newly synthesized for this work as follows.

Rink amide AM LL resin (0.1 mmol) was swelled in dichloro-

methane for 30 min and then rinsed with DMF (365 mL). The

resin was treated with piperidine in DMF (20%, 365 mL). After a

reaction time of 30 min, the solvents were removed by filtration

and the resin was washed with DMF (365 mL), followed by

treatment with Fmoc-D-Ala-OH (155.7 mg, 0.5 mmol) in DMF in

the presence of HATU (mg, 0.5 mmol) and DIPEA (mL). The

reaction progress was monitored by a Kaiser test. After completion

of the coupling, the resin was washed with DMF (365 mL). The

Fmoc protecting group was removed by treatment with piperidine

in DMF (20%, 365 mL, 3610 min). The reaction cycle was

repeated using Fmoc-D-Ala-OH (155.7 mg, 0.5 mmol), Fmoc-

DAP(BOC,tBu)-OH (113.7 mg, 0.2 mmol), Fmoc-D-iso-Gln-OH

(73.7 mg, 0.2 mmol), Fmoc-L-Ala-OH (155.7 mg, 0.5 mmol).

The final Fmoc protecting group was removed by treatment with

piperidine in DMF (20%, 365 mL, 3610 min). The resin was

washed with DMF (365 mL) and the resin bound peptide was

capped by treatment with acetic anhydride and (10%) and DIPEA

(5%) in DMF (365 mL, 3610 min). The resin was washed with

DMF (365 mL), dichloromethane (765 mL), and methanol

(365 mL) and dried in vacuo overnight. The peptide was released

from the resin by treatment with TFA/TIPS/Water (95%/2.5%/

2.5%) in DMF for 2 h. The resin was filtered and washed with

TFA (1610 mL). The filtrate was concentrated under reduced

pressure and co-evaporated with toluene. The crude peptide was

dissolved in water/acetonitrile and purified by semi-preparative

HPLC (Eclipse XDB-C18 column, 5 mm, 9.46250 mm, eluent:

water/acetonitrile/0.1%TFA) to afford, after lyophilization of the

appropriate fraction, the target compound 8. HRMS-MALDI-

TOF calculated for C23H40N8O9Na [M + Na]: 595.29, experi-

mental: 595.41.

Surface plasmon resonance binding assays and kineticanalysis

Binding interactions between ED-PknB and PGN analytes were

examined using a Biacore T100 biosensor system (Biacore Life

Sciences - GE Healthcare). Soluble ED-PknB was immobilized by

standard amine coupling using an amine coupling kit (Biacore).

The surface was activated using freshly mixed N-hydroxysucci-

mide (NHS; 100 mM) and 1-(3-dimethylaminopropyl)-ethylcarbo-

diimide (EDC; 391 mM) (1/1, v/v) in water. Next, ED-PknB

(50 mg/ml) in aqueous NaOAc (10 mM, pH 4.5) was passed over

the chip surface until a ligand density of approximately 5,000 RU

was achieved. The remaining active esters were quenched by

aqueous ethanolamine (1.0 M; pH 8.5). The control flow cell was

activated with NHS and EDC followed by immediate quenching

with ethanolamine. HBS-EP (0.01 M HEPES, 150 mM NaCl,

3 mM EDTA, 0.005% polysorbate 20; pH 7.4) was used as the

running buffer for the immobilization and kinetic studies. Analytes

were dissolved in running buffer and a flow rate of 20 mL/min was

employed for association and dissociation at a constant temper-

ature of 25uC. A double sequential 60 s injection of aqueous

NaOH (50 mM; pH 11.0) at a flow rate of 50 ml/min followed by

5 min stabilization with running buffer was used for regeneration

and achieved prior baseline status. The same experimental surface

was used for approximately 4 weeks and maintained under

running buffer conditions. MTP-DAP (amide/acid) (5) was used as

a positive control in each experiment to check the stability of the

ED-PknB surface activity during the course of the experiments.

To minimize bulk refractive index changes, nonspecific binding

and instrument drift on the generated binding sensorgrams, a

Muropeptides Bind and Localize PknB

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double referencing of the data was performed. First, bulk refractive

index change effects were minimized by preparing all analytes in

the HBS-EP buffer. Then, the binding responses over the

reference surface were subtracted from the active surface to

correct for nonspecific binding. A blank analyte run of running

buffer alone was also subtracted from the specific binding

sensorgrams to minimize instrument noise. Using Biacore T100

evaluation software, the response curves of various analyte

concentrations were globally fitted to the two-state binding model

[40].

Conditioned medium preparationConditioned medium was prepared as previously described

[41]. Briefly, supernatant was obtained from M. tuberculosis H37Rv

culture grown in ADC-supplemented Sauton’s medium containing

0.05% Tween 80 at 37uC with shaking to an optical density at

600 nm (OD600) of 1.2. After centrifugation (4000 rpm, 10 min),

the supernatant was sterilized by passage through 0.22 mm filter,

tested for sterility, and used for the resuscitation experiments.

Dormancy and resuscitation assayTo obtain non-culturable dormant bacilli, Mycobacterium tubercu-

losis H37Rv was grown under long-term oxygen starvation in

broth growth medium (Sauton’s medium containing 0.05% tween

80 and supplemented with ADC) as previously described [10]. In

brief, M. tuberculosis was initially grown to late stationary phase at

37uC with shaking. From this initial culture, 100 mL was

subcultured into 20 ml of growth medium and grown to an

optical density at 600 nm (OD600) of 1.8 to 2. Finally 100 mL was

inoculated into 75 ml of growth medium containing 1.5 mg/ml

methylene blue in a sealed 250-ml flask and grown with shaking at

37uC for 6 or 9 months. Methylene blue became colorless by 10

days of incubation, indicating oxygen depletion.

For resuscitation experiments muropeptides were dissolved in

sterile Sauton’s medium to a concentration of 20 times the binding

constant (KD) of the high affinity compound (6c) The dormant

culture was serially diluted (1021 to 1026) in growth medium.

From each dilution 4 sets of triplicate 100 mL culture were

aliquoted in wells of 96 well plates (one set each for muropeptides

(2 muropeptides tested), growth medium and spent medium).

100 mL of growth medium, muropeptide, or spent medium was

added to each well of the corresponding set. The final

concentration of muropeptide was 10 times the KD of the high

affinity compound, and of the spent medium was 50%. Plates were

incubated at 37uC. Drying was prevented by maintaining sterile

water in outer wells of the plate. After 2 months the wells with

visibly turbid growth were recorded and MPN values were

calculated as previously described [42].

Growth stimulation assayTo investigate the effect of muropeptides on growth initiation of

low inoculum cultures of M. tuberculosis, stationary phase (O.D600

of 2.4–3.6) cultures grown in Sauton’s medium supplemented with

ADC and 0.05% Tween 80 were passed through five micron pore

filter (Millipore) to remove clumps. To obtain a single cell

suspension, the culture was passed five times through a 27K gauge

needle followed by washing three times with Sauton’s medium.

100 ml of a 1024 dilution was inoculated into wells of a 96 well

plate for a final volume of 200 ml. 1x alamar Blue was included in

each well. As above, the final concentration of the muropeptides

was 10 times the KD of the high affinity compound, and of the

spent medium was 50%. Each condition was tested in duplicate.

Growth was monitored in each well by measuring fluorescence

using excitation of 550 nm and emission of 595 nm and plotted as

fluorescence intensity units versus time in days.

MicroscopyFor cellular localization of RFP fusions to intact PknB or its

domains, the corresponding plasmid expressing the fusion under

control of the acetamidase promoter was electroporated into M.

smegmatis cells. The resulting strains were grown in Middlebrook

7H9 liquid medium supplemented with ADC, 0.2% Glycerol and

0.05% Tween 80 to mid-log phase, followed by induction with

0.2% acetamide for 6 hrs. Cells were fixed in 4% paraformalde-

hyde at 37uC for 30 min followed by incubation with 50 mM

ammonium chloride for 5 min at room temperature. Cells were

transferred onto a glass slide, air-dried and one drop of Prolong

Gold antifade reagent (Invitrogen) was applied before covering

with a coverslip. After 24 hrs of curing in the dark, cells were

observed using a Zeiss Axiophot microscope with a 63x differential

interference contrast (DIC) oil immersion objective and red

fluorescence filter. Images were captured by a Spot cooled CCD

camera (Diagnostic Instruments), acquired with Spot software and

processed by Adobe Photoshop CS2.

Western blottingFor cellular localization of native PknB in wild type M.

tuberculosis cells, 60 mg total protein of cytosol, cell wall, cell

membrane and culture filtrate fractions, prepared at Colorado

Statue University and obtained from the Biodefense and Emerging

Infections Research Resources Repository, was fractionated on

10% SDS-PAGE and transferred to a PVDF membrane. The blot

was blocked in Tris-buffered saline containing 0.1% Tween 20

(TBST) and 5% milk for 1 hr at room temperature. The blot was

incubated overnight at 4uC with 1:10,000 dilutions of either a

mouse monoclonal antibody raised against extracytoplasmic

domain of PknB or with a rabbit polyclonal antibody against

PknA. After thorough washing with TBST, the blot was incubated

with a 1:10,000 dilution of HRP-conjugated secondary antibodies

(Cell Signaling) for 3 hrs at room temperature. Finally after 3

washes with TBST the blot was developed with Lumiglo (Cell

Signaling) and the blot image was obtained on a Kodak Image

Station 4000.

Accession numbersPknA: P65726

PknB: POA5S4

Supporting Information

Figure S1 T-Coffee alignment of the four PASTAdomains of M. tuberculosis PknB and the single PASTAdomain of PBP2. Residues/positions corresponding to those

that are conserved in PASTA domains from multiple bacterial

species, according to reference 14, are highlighted in blue.

(TIF)

Figure S2 SDS-PAGE gel showing expression and puri-fication of ED-PknB. M, molecular weight markers: UI, lysate

from uninduced cultures; I, lysate from induced cultures, The

purified protein following removal of the GST tag, shown in the

last lane on the right, was used in the binding experiments.

(TIF)

Figure S3 Sensorgrams of compounds tested in theBiacore binding experiments. The sensorgrams show the

simultaneous concentration-dependent kinetic analysis of two-fold

serial dilutions of each compound. ED-PknB was bound to the

Muropeptides Bind and Localize PknB

PLoS Pathogens | www.plospathogens.org 9 July 2011 | Volume 7 | Issue 7 | e1002182

sensor chip and at time 0 the muropeptide was flowed over the

chip as described in the Materials and Methods section. Positive

deflection of the curve indicates binding in RU (resonance units).

The primary data are shown in red. For compounds that showed

significant binding the data were fitted with a two-state binding

model (black lines) and the corresponding residual values, which

are the signal remaining after the data are fitted to the kinetic

model, are plotted below the sensorgrams. Sensorgrams for

individual muropeptides are shown in A) MTP-Lys (amide) (1);

B) MTrP-Lys (amide) (2a); C) MTrP-Lys (amide) NHAc (2b); D)

MTrP-Lys (Gly) (2c); E) MPP-Lys (D-Ala) (3a); F) MPP-Lys (Gly)

(3b); G) Peptide (amide) (4); H) MTP-DAP (amide/acid) (5); I)

MTrP-DAP (amide/acid) (6a); J) MTrP-DAP (acid/amide) (6b);

K) MTrP-DAP (acid/acid) (6d); L) MTrP-DAP(amide/acid)NHAc

(6e); M) MPP-DAP (amide/acid) (7); N) Peptide (amide/amide) (8).

(PDF)

Figure S4 Immunoblot of subcellular fractions of M.smegmatis showing localization of the RFP-PknB kinasedomain fusion. M. smegmatis was grown to mid log phase,

acetamide was added at a concentration of 0.2% for 8 hours. Cells

were harvested, lysed with a French Press and sub-cellular

fractions were isolated using the protocol developed by the TB

Research Materials Contract at Colorado State University (http://

www.cvmbs.colostate.edu/mip/tb/pdf/scf.pdf). RH615, M. smeg-

matis expressing the RFP-PknB kinase domain fusion under control

of the inducible acetamidase promoter; CM, cytoplasmic mem-

brane fraction; CY, cytoplasm; CW, Cell wall fraction. Though

some fusion protein is present in the cytoplasm, the majority is in

the cell wall and cell membrane fractions, as is native M. smegmatis

PknB.

(TIF)

Figure S5 Live cell imaging of M. smegmatis. Cells

expressing RFP fused to a) full-length PknB, b) to the kinase

domain, linker and transmembrane segment, or c) to ED-PknB

and the transmembrane segment. Fluorescence images are shown

on the left and DIC images on the right. Bar = 1 mm.

(TIF)

Table S1 Kinetic binding parameters for the interaction of

synthetic muropeptides with the PASTA domains of M. tuberculosis

PknB.

(DOC)

Protocol S1 Methods for live cell imaging of M. smegmatis

expressing RFP-PknB fusions.

(DOCX)

Acknowledgments

We thank and Michael Chao and Eric Rubin for the RFP expression

vector, Jessica Wagner and the Harvard Digestive Diseases Center Imaging

Core for assistance with microscopy and Margreet Wolfert for help with

the figures. M. tuberculosis subcellular fractions were obtained through

NIAID Contract No. HHSN266200400091C, entitled "Tuberculosis

Vaccine Testing and Research Materials," awarded to Colorado State

University. Antibodies to PknA were obtained from Vertex Pharmaceu-

ticals Incorporated.

Author Contributions

Conceived and designed the experiments: MM JA G-JB RNH. Performed

the experiments: MM JA XL JC. Analyzed the data: MM JA XL JC G-JB

RNH. Contributed reagents/materials/analysis tools: MM RNH G-JB.

Wrote the paper: MM JA G-JB RNH. Revision of manuscript: MM G-JB

RNH.

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